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Team:EPF Lausanne/Envelope stress responsive bacteria
From 2014.igem.org
Envelope Stress Responsive Bacteria
The pathway we engineered in Bacteria is the Cpx two component regulatory system. It's natural function is to control the expression of "survival" genes whose products act in the periplasm to maintain membrane integrity. This ensures continued bacterial growth even in environments with harmful extractoplasmic stresses. The Cpx two component regulatory system belongs to the class I histidine kinases and includes three main protein (3 blocks with the descriptions of the proteins) (pathway draw)
Split reporter proteins: Infrared Fluorescent Protein
Solving the orientation of CpxR homodimerization: Split IFP
As the orientation of CpxR homodimerization is not very well studied, we had to resolve which end (C or N terminal) of the CpxR would be the most suitable for the fusion of the IFP fragments. We designed the four following constructs:
Both IFP[1] and IFP[2] at the C terminal of CpxR
IFP[1] at the C terminal and IFP[2] at the N terminal
IFP[1] at the N terminal and IFP[2] at the C terminal
Both IFP[1] and IFP[2] at the N terminal of the CpxR
Procedure
The first experiment was achieved on a plate reader in order to measure the signal of the four different strains under different stresses: KCL, cupper, KOH or silica beads, which are thought to activate the pathway (link). We also measured as negative control the signal of strains expressing one part of the split only (IFP[1]-CpxR or IFP[2]-CpxR). Three measurements were necessary to finally conclude that only the first configuration works, when both split part of IFP are at the C terminal of the CpxR. GRAPH
What could be the other sources of stress activating the pathway
Antibiotics hypothesis
AFM pictures
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